Modification of Lysine Residues of Horseradish Peroxidaseand Its Effect on Stability and Structure of the Enzyme

Leila Hassani & Rasool Nourozi

Received: 1 October 2013 /Accepted: 21 January 2014# Springer Science+Business Media New York 2014

Abstract Biotechnology is consistently seeking improved enzyme stability. Enzymes havegreat properties, although their marginal stability limits their applications. Among the strate-gies for improving stability of the enzymes, chemical modification is a simple and effectivetechnique. In the present study, chemical modification of horseradish peroxidase (HRP) wascarried out with 2,3-dichloromaleic anhydride and 2,3-dimethylmaleic anhydride. HRP is animportant heme-containing enzyme. It is widely applied in pharmacological, chemical, andmedical industries. Here, thermal stability of HRP was investigated at different temperatures.In addition, the enzyme stability was evaluated in urea, DMSO, alkaline pH, and hydrogenperoxide solutions by spectroscopic techniques. Structural investigation indicated that the bothanhydrides slightly decrease compactness of the enzyme structure. The results also indicatedthat 2,3-dichloromaleic anhydride increases thermal stability of the enzyme and its stability inurea and DMSO solutions, but 2,3-dimethylmaleic anhydride only stabilizes HRP in ureasolution. Furthermore, the experiments implied that none of the modifiers are effective on thestability of HRP in extreme pH and oxidative condition. Catalytic efficiency and activationenergy did not change remarkably following reaction of the enzyme with the both carboxylicanhydrides. Consequently, improvement in the stability of HRP depends on not only the typeof modifier but also denaturing condition.

Keywords Horseradish peroxidase . Chemical modification . 2,3-Dichloromaleic anhydride .

2,3-Dimethylmaleic anhydride . Spectroscopy

Introduction

Biotechnology is consistently seeking improved enzyme stability. Enzymes are cata-lysts, which have great properties like high specificity, selectivity, and biodegradabil-ity, although the relatively short lifetime of enzymes limits their medical and industrial

Appl Biochem BiotechnolDOI 10.1007/s12010-014-0756-y

Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-0756-y)contains supplementary material, which is available to authorized users.

L. Hassani (*) : R. NouroziDepartment of Biological Sciences, Institute for Advanced Studies in Basic Sciences (IASBS),P.O. Box 45195-1159, Gava-zang, Zanjan 45137-6731, Irane-mail: hasani@iasbs.ac.ir

applications. Improvement in enzymes stability prolongs their lifetime and thereforereduces the cost of these biocatalysts [1, 2].

There are different approaches to improve enzymes stability in non-conventionalmedium including isolation of stable enzymes from extremophiles, protein engineer-ing, immobilization, chemical modification, and solvent engineering [1, 3]. Amongdifferent approaches, chemical modification provides a rapid and inexpensive methodto stabilize enzymes by cross-linking or introduction of monomeric or polymericmoieties. In spite of the development of modern techniques for stabilization ofenzymes, chemical modification is still applicable [4].

It has been reported in literature that stability and activity of enzymes change dueto covalent modification of protein surface with hydrophilic low-molecular weightgroups. Such a modification results in holding the hydration shell of protein andincreasing protein resistance to denaturing condition [5–9].

Here, horseradish peroxidase (HRP, donor: hydrogen peroxide oxidoreductase,E.C. 1.11.1.7) has been modified with 2,3-dichloromaleic anhydride and 2,3-dimethylmaleic anhydride (Scheme 1), and the effect of modifiers on stability andstructure of the enzyme has been investigated. HRP is an important heme-containingenzyme. It has been studied for more than a century. HRP uses hydrogen peroxideas electron acceptor for oxidation of a wide variety of substrates. This enzyme hasmany industrial and medical applications; for example, it is used as a reagent fororganic synthesis, biotransformation, and treatment of waste waters. HRP is alsocommonly used in techniques such as ELISA and immunohistochemistry [10, 11].

We previously modified HRP using citraconic anhydride, trimellitic anhydride,and pyromellitic anhydride introducing one, two, and three carboxylic groups pereach lysine residues, respectively. All modifiers improve thermal stability of HRP.The effect of dicarboxylic anhydride on the thermal stability of HRP was more thanthat of monocarboxylic anhydride, but the monocarboxylic anhydride was effectivein wider temperature range in comparison with the dicarboxylic anhydride. Weexplained the improved thermostability based on hydrophilization and concluded thathydrophilic groups introduced on the surface of HRP improve its thermal stability[12–14]. In this investigation, HRP was modified with the two other modifiers inorder to understand more about the effect of modification of the protein surface onits stability under non-conventional condition. 2,3-Dichloromaleic anhydride and 2,3-dimethylmaleic anhydride respectively introduce two CH3 and two Cl on the surfaceof enzyme. Thermal stability and stability of the enzyme in the organic solvent,alkaline pH, and hydrogen peroxide as oxidative agent were evaluated by thespectroscopic techniques. In addition, the effect of chemical modification on thestructure of HRP was studied by circular dichroism and fluorescence spectroscopy.

Scheme 1 Structure of 2,3-dichloromaleic anhydride and 2,3-dimethylmaleic anhydride

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Material and Methods

Materials

2,3-Dichloromaleic anhydride, 2,3-dimethylmaleic anhydride, 4-aminoantipyrine,trinitrobenzene sulfonic acid (TNBSA), and horseradish peroxidase (P6782) were obtainedfrom Sigma.

Chemical Modification of HRP with 2,3-Dichloromaleic Anhydride and 2,3-DimethylmaleicAnhydride

Chemical modification of HRP using 2,3-dichloromaleic anhydride and 2,3-dimethylmaleic anhydride was performed according to the procedure described byDixon and Perham [15]. The two carboxylic anhydrides were separately dissolved inDMSO at the highest possible concentration and added dropwise to 1.5 mL volume ofthe enzyme (4.5 mg/mL) prepared in 100 mM borate buffer, pH 8. Addition of theanhydride brought about a drop in pH; therefore, the pH of the stirred solution waskept constant by adding small amount of 1 M NaOH. The reaction was completedafter addition of 50 μL of the modifier. Then, the reaction mixture was kept for 1 hat room temperature, and the enzyme was subsequently separated from the freemodifier by dialysis against 20 mM phosphate buffer, pH 7. In control experiments,the protein whose lysine residues had not been modified was treated in the same way.

Determination of the Number of Modified Lysine Residues

To determine the number of modified lysine residues, the number of free amino groups wasmeasured using TNBSA assay [16–18]. HRP was dissolved in 0.1 M sodium bicarbonate,pH 8.5, and 250 μL of 0.1 % TNBS was added to 500 μL of 200 μg/mL of HRP, and thesamples were incubated at 37 °C for 2 h. Then, 250 μL of 10 % sodium dodecyl sulfate (SDS)and 125 μL of 1 N HCl were added to the samples. Subsequently, absorbance of the sampleswas measured at 335 nm. The percentage of modified amino groups was calculated by thefollowing equation:

Modified amino groups %ð Þ ¼ 1−Am

Ac

� �� 100

where Am and Ac are, respectively, the absorbance of non-modified and modified samples at335 nm.

Determination of the Protein Concentration, Kinetic Parameters, and Activation Energyof the Enzymatic Reaction

Concentration of HRP was measured using the molar extinction coefficient of 102/mM/cm at403 nm.

The enzyme was assayed by colorimetric method using 4-aminoantipyrine andhydrogen peroxide as color-generating substrates [19]. A reaction mixture containing0.17 M phenol, 0.0025 M 4-aminoantipyrine, and 0.0017 M hydrogen peroxide in atotal volume of 600 μL in phosphate buffer 0.1 M was prepared. Then, 50 μL ofHRP (0.002 mg/ml) was added to 600 μL of the substrates solution, and after 3 s, the

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rate of color generation at 510 nm was measured by UV M501-Camspect spectro-photometer. The rate of reaction was determined using the following equation:

VUnit

mg

� �¼ ΔA510=min

6:58�mg enzyme=reaction mixture

For determination of Vmax (maximum rate of enzymatic reaction) and Km (Michaelisconstant) using Lineweaver–Burk plot [20], the enzyme was assayed in the range of 0.026–0. 85 mM H2O2.

To calculate the activation energy, the rate of enzymatic reaction was determined at thetemperature range of 15–35 °C.

Determination of the Enzyme Stability on the Denaturing Condition

The effect of temperature on the rate of enzymatic reaction was determined over a temperaturerange of 50–80 °C. At first, the enzyme was dissolved in 20 mM phosphate buffer, pH 7, andits final concentration was adjusted to 0.002 mg/mL. Subsequently, the samples were incu-bated at the selected temperatures. Then, the samples were removed at the defined timeintervals and cooled on ice. After they were cooled, their remaining activity was determined.The activity of the same enzyme solution, kept on ice, was considered as the control (100 %).

To evaluate the effect of pH on activity of the non-modified and modified HRP, 0.002 mg/mL of the enzyme was prepared in the pH range of 7–11.

In order to investigate the effect of chemical modification on stability of the enzyme in theorganic solvent and the chemical denaturant, the enzyme was prepared in 20 mM tris buffer,pH 7, containing 1–8 M urea and 0–70 % DMSO. After 1 h incubation at room temperature,the enzyme samples were added to the substrate solution dissolved in phosphate buffer, pH 7,and the rate of reaction was measured.

The enzyme was also assayed at the high concentrations of H2O2 to evaluate the effect ofoxidative condition on the rate of enzymatic reaction. It is necessary to note that the optimumconcentration of H2O2 as substrate is 0.85 mM; at the higher concentration of H2O2, theactivity of the enzyme decreases, because H2O2 oxidizes the enzyme. Accordingly, we assayedthe enzyme in 0.85–3.4 mM of H2O2 to evaluate the effect of chemical modification onstability of the enzyme on the oxidative condition.

Circular Dichroism

Circular dichroism (CD) measurements were performed on JASCO J-810 circular dichroismspectrometer at 25 °C. Far-UV CD spectra were registered in a quartz cell of 1-mm pathlength. The spectra of near-UV and visible regions were obtained in a 1-cm cell. In allmeasurements, the protein concentration was 0.2 mg/mL. The scans were recorded using abandwidth of 1 nm. The molar ellipticity was determined as [θ]=(θ×100MRW)/(cl), where c isthe protein concentration in milligram per milliliter, l is the light path length in centimeters,MRW is mean residue weight of HRP, and θ is the measured ellipticity in degrees [21].

Fluorescence Spectroscopy

Intrinsic fluorescence of the protein was measured at room temperature using Varian CaryEclipse fluorescence spectrophotometer in a quartz cell of 1-cm path length in 20 mM

Appl Biochem Biotechnol

phosphate buffer, pH 7. The excitation wavelength was separately set at 280 and 295 nm.Concentration of the protein was 0.05 mg/mL.

Fluorescence quenching was carried out by addition of acrylamide to the protein solution.The excitation wavelength was 295 nm for the quenching experiment.

Results

The Number of Modified Lysine Residues

The result of TNBSA assay implied that 52 and 41 % of the amino groups of HRPare, respectively, modified by 2,3-dichloromaleic anhydride and 2,3-dimethylmaleicanhydride. HRP has six lysine residues (Lys-65, Lys-84, Lys-149, Lys-174, Lys-232,and Lys-241); consequently, 3.1 and 2.5 of the six lysine residues bind to themodifiers.

Effect of the Chemical Modification on Stability of HRP Under the Different DenaturingConditions

As noted, thermal stability of HRP was evaluated over a temperature range of 50–80 °C. In theprevious report [12], we concluded that the effect of chemical modification with carboxylicanhydrides on thermal stability of HRP depends on the incubated temperature. It was shownthat behavior of the modified enzyme at 70–80 °C is different from that at the lowertemperatures (50–60 °C). For example, chemical modification of HRP with trimellitic anhy-dride results in a considerable enhancement of the thermal stability at the lower temperatures,but the same modification causes to decrease of the thermal stability at the higher temperatures.Accordingly, in this investigation, relatively wide range of temperatures was selected tocompare thermoinactivation of the non-modified form of HRP with the modified forms. Theresults are shown in Fig. 1 and supplementary data (Figs. 1S–5S). Half-life of inactivation (t1/2)was also presented in the inset of Fig. 1. Half-life of inactivation is the time it takes the activityof the protein to be reduced by half, following incubation under specific condition. As shownin Fig. 1a, the chemical modification with 2,3-dichloromaleic anhydride leads to a remarkabledecrease in the rate of thermoinactivation, but 2,3-dimethylmaleic anhydride as a modifier hasno significant effect on the thermal stability of HRP at 55 °C. At 55 °C, 2,3-dichloromaleicanhydride modified form of HRP is more stable than the non-modified enzyme, although 2,3-dimethylmaleic anhydride modified form loses its activity faster than the non-modified form(supplementary data, Fig. 1S). Similar results were obtained at 60 °C (Fig. 2b). Half-life ofinactivation for dichloromaleic anhydride modified form is, respectively, 1.9- and 4.7-foldmore than that of the non-modified form at 55 and 60 °C.

Dichloromaleic anhydride succeeds in stabilizing HRP at 65 °C, and dimethylmaleicanhydride slightly increases the thermal stability following incubation of the enzyme for 3and 5 h at 65 °C.

At the higher temperatures, i.e., 70, 75, and 80 °C, no remarkable change is observed in thethermal stability of HRP upon modification of the enzyme with the both modifiers (supple-mentary data, Figs. 3S–5S).

Taken together, the results indicated that the reaction of HRP with dichloromaleic anhy-dride leads to improve thermal stability of HRP at the temperatures below 70 °C, although theenzyme gains no stability or loses the thermal stability, when it is modified withdimethylmaleic anhydride.

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As noted, besides thermal stability, the enzyme stability was evaluated on the otherdenaturing conditions. Figure 2a shows remaining activity of the non-modified and modifiedforms of HRP at different concentrations of urea. As shown in Fig. 2a, remaining activity ofthe enzyme increases due to modification with dichloromaleic anhydride and dimethylmaleicanhydride, indicating that the both modifiers improve stability of HRP. For example, at 1 Murea, the non-modified enzyme loses 82 % of its initial activity, whereas the lost activity ofdichloromaleic anhydride and dimethylmaleic anhydride modified forms of HRP is, respec-tively, 48 and 44 %.

The remaining activity of the non-modified HRP was compared with the modifiedones in different concentrations of DMSO in Fig. 2b. Obviously, dichloromaleicanhydride modified form is slightly more stable than the non-modified HRP, but theother modified form is slightly less stable than the non-modified form on the samecondition.

Overall, the effect of the modifiers on the stability of the enzyme in the organic solvent isnot pronounced.

The stability of HRP at extremes of pH and oxidative condition was also evaluated in ourinvestigation. The results (supplementary data, Figs. 6S and 7S) indicated no significantdifference between the non-modified and the both modified forms of HRP at extremes neitherof pH nor on the oxidative condition.

Fig. 1 Irreversible thermoinactivation and half-life of thermoinactivation (t1/2) (inset) of non-modified,dichloromaleic anhydride (Cl) and dimethylmaleic anhydride (CH3) modified forms of HRP at 50 °C (a) and60 °C (b), pH 7.0

Fig. 2 Remaining activity of non-modified, dichloromaleic anhydride (Cl) and dimethylmaleic anhydride (CH3)modified forms of HRP in urea (a) and DMSO (b) solution

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The Effect of Chemical Modification on Activation Energy and Catalytic Efficiencyof the Enzyme

The results (supplementary data, Figs. 8S and 9S) indicated that Arrhenius and Lineweaver–Burk plots of the enzyme are linear. The values of Ea, Vmax, Kcat, Km, and kcat/Km (catalyticefficiency) are presented in Table 1. As shown, the mentioned parameters do not changeremarkably, after chemical modification of the enzyme with the both modifiers; only a slightincrease in the value of Vmax, kcat, and catalytic efficiency of the enzyme is observed due to thechemical modifications.

Circular Dichroism Measurements

Far-UV CD spectrum of HRP (Fig. 10S, supplementary data) has negative band at about 222and 208 nm, indicating α-helical structure of the enzyme [22]. A very little decrease is shownin the intensity of far-UV spectrum of HRP after modification with the both modifiers. Similarresult was obtained in near-UV region. As shown in Fig. 3a, the chemical modifications causeto a decrease in the intensity of negative and positive bands at about 280 and 310 nm. Inaddition, induced CD signal at 403 nm (Fig. 3b) slightly decreases upon the chemicalmodification.

Fluorescence Spectroscopy

Figure 4 shows intrinsic fluorescence spectra of the non-modified and modified forms of HRPat room temperature with excitation at 295 nm. As is well known, the fluorescence emissionexcited at 295 nm arises solely from tryptophan residues. As shown, the intensity of Trpemission decreases after chemical modification of the enzyme by dichloromaleic anhydrideand dimethylmaleic anhydride.

It is necessary to note that the intensity of emission decreased after the chemical modifi-cations, when the enzyme was excited at 280 nm (supplementary data, Fig. 11S).

Quenching of Trp by acrylamide was used to measure Trp depth. Stern–Volmer plots(Fig. 12S) indicate the difference between the non-modified and modified forms of HRP. Asshown, the slope increases after the chemical modification, indicating Trp residue of HRP ismore exposed to the solvent after the chemical modification.

Discussion

As noted, in this investigation, lysine residues of HRP were chemically modified with twocarboxylic anhydrides including dichloromaleic anhydride and dimethylmaleic anhydride.

Table 1 Kinetic parameters and activation energy for the enzymatic reaction of non-modified dichloromaleicanhydride (Modified 1) and dimethylmaleic anhydride (Modified 2) modified forms of HRP

Vmax (U/mg) Km (mM) kcat (s−1) kcat/Km (s−1/mM) Ea (kCal/mol)

Non-modified 589±25 0.28±0.032 442±18 1,578 19. 07±2.04

Modified 1 632±16 0.29±0.007 474±1 1,634 18.81±0.46

Modified 2 646±29 0.27±0.01 484±2 1,792 18.85±0.25

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As mentioned in the results, about half of lysine residues of HRP were modified with themodifiers. This result agrees with our previous investigations. We reported that about threelysine residues of HRP are modified due to the chemical modification with citraconicanhydride, trimellitic anhydride, and pyromellitic anhydride [12–14]. Since only three of thesix lysine residues of HRP are surface accessible [14], it is very likely that most of the surfaceaccessible lysine residues of HRP are chemically modified by the anhydrides. This probabilityagrees with the result of O’Brien and Fagain [23]. They modified HRP with bifunctionalcompound EGNHS and identified the location of modified lysine residues by proteolyticfragmentation, peptide sequencing, and mass spectrometry techniques. All three methodsindicated that the chemical modification leads to complete modification of Lys-232, partialmodification of Lys-174 and Lys-241, and very little reaction of Lys-65, Lys-84, and Lys-149.Accordingly, although we need more investigations, it is reasonable to predict that the threesurface accessible residues are most likely to change due to the modification.

Fig. 3 Near-UV (a) and soret (b) circular dichroism spectra of non-modified, dichloromaleic anhydride (Cl) anddimethylmaleic anhydride (CH3) modified forms of HRP at room temperature and pH 7.0

Fig. 4 Trp emission fluorescence spectra of non-modified (1) dichloromaleic anhydride (2) and dimethylmaleicanhydride (3) modified forms of HRP excited at 295 nm at room temperature and pH 7.0

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Circular dichroism indicated that the intensity of near-UV and visible spectra of HRPslightly decreases upon the chemical modification. Circular dichroism spectroscopy givesinformation about structure of the protein. The secondary and tertiary structures of the proteincan be respectively evaluated by CD spectroscopy in the far-UV (190–250 nm) and near-UV(250–320 nm) spectral regions. In addition, non-protein cofactors have absorbance over a widespectral range [22]. For example, heme group of HRP has absorbance in the soret region.Heme shows no CD signals, when it is free in solution, but, only when bound to the protein,shows induced CD signal used to give structural information about the catalytic center of HRP[14, 22]. The magnitude of CD spectra of a protein depends on the rigidity of the protein, withthe more highly mobile side chains having lower intensities [22]; therefore, according to theCD results, it can be concluded that compactness of the tertiary structure and the heme pocketof HRP decrease upon the modification with dichloromaleic anhydride and dimethylmaleicanhydride.

As noted, intensity of the intrinsic fluorescence spectra decreases after the chemicalmodification. Fluorescence spectroscopy can be used to study conformational changes ofproteins. Fluorescence emission of proteins is sensitive to polarity of medium. In a hydropho-bic environment, tyrosine and tryptophan residues have high quantum yield. In contrast, inhydrophilic environment, their quantum yield decreases leading to low fluorescence intensity[24]. A decrease in fluorescence emission may also occur due to the proximity of a quenchersuch as heme group to Trp residue [25–27].

HRP contains one Trp residue, Trp-117, and five tyrosine residues [28]. Trp-117locates between two α-helices at the side opposite the entrance to the heme-bindingpocket [29]. Accordingly, a possible reason for the fluorescence result is exposure ofthe only Trp residue of HRP to the solvent or polar environment. The observeddecrease in the fluorescence emission may also occur, because of proximity of theheme group to the tryptophan residue [25–27]. As noted, the quenching experimentindicated that Trp residue of HRP is more exposed to the solvent after the chemicalmodification. Thus, it can be concluded that the intrinsic fluorescence intensitydecreases because of exposure of Trp residue to the solvent. Probably, distancebetween the heme group and the Trp-117 is another important factor for the fluores-cence result. As noted, the results of circular dichroism indicated a little decrease incompactness of the tertiary structure of HRP around the heme group. This changepresumably affects the relative orientation or distances between the heme and the Trp-117 residue.

As mentioned, dichloromaleic anhydride increases thermal stability of HRP at thetemperatures below temperature midpoint (tm) of HRP, that is, 77 °C [14]. On theother hand, this modifier stabilizes HRP, when less than half of the enzyme structurethermally unfolds. It was previously reported that negatively charged residues arelocated near amino groups of Lys-174, Lys-232, and Lys-241. Therefore, it can beconcluded that modification of HRP with dichloromaleic anhydride, which reversesthe charges of these lysine residues, would be electrostatically unfavorable for theprotein stability at the high temperatures [30].

The results indicated that catalytic efficiency of the enzyme does not changeconsiderably and structure of the enzyme slightly changes upon chemical modificationwith the both modifiers, although the modifiers affect the stability of the enzyme. Inspite of the fact that both modifiers have no effect on the stability of HRP on somedenaturing conditions, taken together, dichloromaleic anhydride improves the stability,but dimethylmaleic anhydride fails to stabilize the enzyme in some conditions anddecreases the stability on the other conditions.

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Although it is not possible to explain the observed changes solely based on the introductionof additional hydrophobe and hydrophile groups onto the surface of HRP, it is most probablethat the change in the surface influences the stability.

Carboxylic anhydrides (X-CO-R) can chemically modify amino groups (NH2) of theprotein (P). The reaction occurs as follows [31]:

P−NH2 þ X−CO−R→P−NH−CO−Rþ HX

2,3-Dichloromaleic anhydride and 2,3-dimethylmaleic anhydride as the modifiers respec-tively introduce two CH3 and two Cl on the surface of the enzyme. The positive charges of theamino groups are eliminated, and negatively charged carboxylates are introduced upon thechemical modification of the protein with the anhydrides. In addition, 2,3-dichloromaleicanhydride and 2,3-dimethylmaleic anhydride introduce two CH3 and two Cl on the surfaceof the enzyme.

The protein surface is primarily responsible for interaction with the solvent, so theproperties of the protein surface may be important for stabilizing the protein on denaturingcondition [6]. Hydrophilized surface not only keeps hydration shell but also forms additionalelectrostatic interaction with solvent. In contrast, contacts of nonpolar groups with water arethermodynamically unfavorable [6, 32, 33]. Accordingly, it is most probable that hydrophilicand hydrophobic features of 2,3-dichloromaleic anhydride and 2,3-dimethylmaleic anhydrideare important factors in the enzyme stability.

It is necessary to remember that neither 2,3-dichloromaleic anhydride leads to increasingthe stability of HRP in the all conditions nor 2,3-dichloromaleic anhydride leads to decreasingthe stability in the all cases. As noted, modification of HRP with 2,3-dichloromaleic anhydrideresults in increasing of the stability in urea solution, but on the other denaturing conditions, thestability does not change or decreases upon the same modification. Furthermore, in the case of2,3-dichloromaleic anhydride, the stability of HRP increases at the temperature below 65 °C,in urea solution and slightly in DMSO. However, 2,3-dichloromaleic anhydride has no effecton the stability of HRP in oxidative condition and alkaline pH. Consequently, hydrophilizationand hydrophobization of the surface of enzyme are not the only reason for stability of HRP;certainly, other factors influence the stability of the enzyme.

Conclusion

Modification of lysine residues of HRP with the two anhydrides was shown in the presentwork. Unlike 2,3-dimethylmaleic anhydride, 2,3-dichloromaleic anhydride has a favorableeffect on the stability of HRP. As well known, protein instability is main roadblocks to thesuccessful application of the enzymes. HRP has good stability characteristics [34, 35],contributing to its widespread use. Any worthwhile biosensor or bioreactor involving HRP,however, must be robust and stable in operation. The enzyme must function reliably undervarying conditions over an extended period. Its intrinsic stability characteristics may not besufficient to cope with these demands, and improvement of the stability increases its potentialapplication range [34, 36, 37].

Several studies have tried to increase the stability of HRP by modern approaches. In somecases, the protein exhibited greater stability but lower catalytic efficiency after immobilizationand site-directed mutagenesis [38–41]. Here, it was shown that stability of HRP increases witha simple and low-cost traditional method with no significant change in the structure andcatalytic efficiency of the enzyme. In addition, in the most cases, the stability of modified

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forms of HRP was investigated only at high temperatures, extreme pH, and/or organic solvents[12–14, 42], but in this investigation, stability of the enzyme in hydrogen peroxide andchemical denaturant solutions was evaluated.

On the whole, it was shown that improvement in the stability of protein due to chemicalmodification depends on not only the feature of modifiers but also denaturing conditions. Amodifier may stabilize the enzyme on some denaturing conditions but fail to stabilize it or hasno effect on the stability on the other denaturing conditions.

Acknowledgments Financial support for this work was provided by Research Council of Institute for Ad-vanced Studies in Basic Sciences.

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